Morphology and function of human Leydig cells in vitro. Immunocytochemical and radioimmunological analyses

The aim of our study was to show whether the cells isolated from testes of patients underwent bilateral orchiectomy for prostatic cancer are able to grown in vitro, and if so, are functionally active. Immuncytochemistry was performed to show the functional status of human cultured cells. In detail, immunolocalization of luteinizing hormone receptors (LHR), mitochondria, and cytoskeletal elements was demonstrated. Moreover, radioimmunological assay was used to measure testosterone secretion by cultured Leydig cells. Using Nomarski interference contrast and fine immunofluorescence analysis the positive immunostaining for LHR was observed in almost all Leydig cells, however it was of various intensity in individual cells. Testosterone measurement revealed significant difference between testosterone secretion by hCG-stimulated and unstimulated Leydig cells (p<0.05). Moreover, testosterone levels were significantly higher in 24- and 48-hour-cultures than in those of 72 hrs (p<0.05). Morphological analysis of Leydig cells in culture revealed the presence of mononuclear and multinucleate cells. The latter cells occurred in both hCG-stimulated and unstimulated cultures. In Leydig cells labeled with a molecular marker MitoTtracker, an abundance of mitochondria and typical distribution of microtubules and microfilaments were observed irrespective of the number of nuclei within the cell, suggesting no functional differences between mono- and multinucleate human Leydig cells in vitro. Since the percentage of multinucleate cells was similar in both hCG-stimulated and unstimulated cultures (23.70% and 22.80%), respectively, the appearance of these cell population seems to be independent of hormonal stimulation

Variations in popliteal fossa venous anatomy: implications for diagnosis of deep-vein thrombosis

Background: To retrospectively review the bilateral venous system within the popliteal fossa to evaluate the types of variations and their frequency seen in venous anatomy.Materials and methods: During routine dissection of formalin-fixed cadavers, a retrospective review of 32 bilateral (64 limbs) lower limbs obtained from adult donors was performed. Deep veins present in the popliteal fossa were evaluated according to predetermined criteria for the presence of duplication of vessels and interindividual variations in venous anatomy.Results: More than one deep venous vessel was seen in the popliteal fossa in 20 (31.3%) of 64 limbs. In 12 (18.7%) cases there was a high (just below the level of the adductor hiatus) origin of the popliteal vein: from 2 tributaries in 10 (15.6%) and 3 tributaries in 2 (3.1%). In 5 (7.8%) cases true duplicated popliteal veins were observed. There were also 3 (4.7%) cases, including one bilateral, of persistent sciatic vein.Conclusions: Variations in popliteal fossa venous anatomy are common and have important implications for the diagnosis of deep vein thrombosis

Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display.

A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties

Bacterial expression and secretion of various single-chain Fv genes encoding proteins specific for a Salmonella serotype B O-antigen.

Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and VL-VH orientation on expression, secretion, and binding to a Salmonella O-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS)2 sequence. VL and VH domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were approximately 50 mg/liter, chiefly in the form of inclusion bodies. With VL in the NH2-terminal position, the amount of secreted product obtained was 2.4 mg/liter, but when VH occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG

Progress in plasma research at IPJ and IPPLM, Poland

The most important results of theoretical and experimental studies of plasmas, which have been achieved at the IPJ in Swierk and IPPLM in Warsaw recently, are presented. Studies of physical phenomena in PF discharges, development of diagnostic techniques and research on new plasma technologies, as performed at IPJ, have been summarized. Studies of dense magnetized plasmas, investigation of physics and applications of laser-produced plasmas; and research on the development of advanced diagnostic techniques for the EURATOM fusion program, as performed at IPPLM, are also described

Concerted Phenotypic Flexibility of Avian Erythrocyte Size and Number in Response to Dietary Anthocyanin Supplementation

Background: Endurance flight impose substantial oxidative costs on the avian oxygen delivery system. In particular, the accumulation of irreversible damage in red blood cells can reduce the capacity of blood to transport oxygen and limit aerobic performance. Many songbirds consume large amounts of anthocyanin-rich fruit, which is hypothesized to reduce oxidative costs, enhance post-flight regeneration, and enable greater aerobic capacity. While their antioxidant benefits appear most straightforward, the effects of anthocyanins on blood composition remain so far unknown. We fed thirty hand-raised European starlings (Sturnus vulgaris) two semisynthetic diets (with or without anthocyanin supplement) and manipulated the extent of flight activity in a wind tunnel (daily flying or non-flying for over two weeks) to test for their interactive effects on functionally important haematological variables. Results: Supplemented birds had on average 15% more and 4% smaller red blood cells compared to non-supplemented individuals and these diet effects were independent of flight manipulation. Haemoglobin content was 7% higher in non-supplemented flying birds compared to non-flying birds, while similar haemoglobin content was observed among supplemented birds that were flown or not. Neither diet nor flight activity influenced haematocrit. Conclusion: The concerted adjustments suggest that supplementation generally improved antioxidant protection in blood, which could prevent the excess removal of cells from the bloodstream and may have several implications on the oxygen delivery system, including improved gas exchange and blood flow. The flexible haematological response to dietary anthocyanins may also suggest that free-ranging species preferentially consume anthocyanin-rich fruits for their natural blood doping, oxygen delivery-enhancement effects

Catalytic cracking performance of alkaline-treated zeolite Beta in the terms of acid sites properties and their accessibility

The zeolite Beta is considered as a promising additive for FCC catalyst in diesel oil production. In this article, it is shown that hierarchical zeolite Beta obtained by an optimized desilication procedure increases diesel and propylene yields during gas oil cracking reaction. The alkaline treatment of zeolite Beta (Si/Al = 22) by desilication with NaOH and NaOH&TBAOH was investigated. The catalytic performance improvement of desilicated zeolite Beta has been rationalized by deep characterization of the samples including X-ray diffraction, low-temperature adsorption of nitrogen, solid-state 29Si MAS NMR and IR studies of acidity. Finally, the catalytic performance of the zeolites Beta was evaluated in the cracking of n-decane, 1,3,5-tri-iso-propylbenzene, and vacuum gas oil. It was found that desilication with NaOH&TBAOH ensures the more uniform intracrystalline mesoporosity with the formation of narrower mesopores, while preserving full crystallinity resulting in catalysts with the most appropriated acidity and then with better catalytic performance.Also, M.C.I.L., J.M.T. and F.R. thank financial support from the Spanish Ministry of Economy and Competitiveness through the Severo Ochoa program (SEV-2012-0267) as well as operating grants Consolider Ingenio Multicat (CSD-2009-00050) and MAT-2012-3856-0O2-01.Tarach, K.; Gora-Marek, K.; Tekla, J.; Brylewska, K.; Datka, J.; Mlekodaj, K.; Makowski, W.... (2014). Catalytic cracking performance of alkaline-treated zeolite Beta in the terms of acid sites properties and their accessibility. Journal of Catalysis. 312:46-57. https://doi.org/10.1016/j.jcat.2014.01.009S465731

Dynamics of hormonal disorders following unilateral orchiectomy for a testicular tumor

Testicular tumors and their treatment interfere with homeostasis, hormonal status included. The aim of the study was to evaluate hormonal disorders of the pituitary–gonadal axis in men treated for testicular tumors. One hundred twenty-eight men treated for a unilateral testicular tumor at our institution were included. The hormonal status was prospectively evaluated in 62 patients before orchiectomy, 120 patients 1 month after orchiectomy and 110 patients at least 1 year after the treatment. The concentrations of human chorionic gonadotropin (hCG), testosterone (T), estradiol, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured. The clinically significant testosterone deficiency was defined either as testosterone <2.31 ng/mL or testosterone within the range of 2.31–3.46 ng/mL but simultaneous with T/LH ratio ≤1. Changes in hormone levels were significant: LH and FSH rose in the course of observation, and the concentration of hCG, testosterone, estradiol decreased. PRL concentration was the lowest at 1 month after orchiectomy. In multivariate analysis, the risk of the clinically significant testosterone deficiency was 0.2107 (95% CI 0.1206–0.3419) prior to orchiectomy, 0.3894 (95% CI 0.2983–0.4889) 1 month after surgery and 0.4972 (95% CI 0.3951–0.5995) 1 year after the treatment. The estradiol concentration was elevated in 40% of patients with recently diagnosed testicular cancer and that was correlated with a higher risk of testosterone deficiency after the treatment completion. Hormonal disorders of the pituitary–gonadal axis in men treated for testicular tumors are frequent. The malignant tissue triggers paraneoplastic disorders that additionally disturb the hormonal equilibrium

Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant

We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS) expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE2 levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO) and N-acetyl-D-glucosaminidase (NAG) activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE2 and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process